Role of toll-like receptor 4 in skeletal muscle damage in chronic limb-threatening ischemia.

ABSTRACT

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors in the innate immune system. In particular, the TLR4-mediated immune response has been implicated in ischemia-induced tissue injury. Mounting evidence supports a detrimental role of the innate immune system in the pathophysiology of skeletal muscle damage in patients with chronic limb-threatening ischemia (CLTI), in whom patient-oriented functional outcomes are poor. The overall aim of this study was to investigate the potential role of TLR4 in skeletal muscle dysfunction and damage in CLTI. Methods: The role of TLR4 in ischemic muscle was investigated by (1) studying TLR4 expression and distribution in human gastrocnemius muscle biopsies, (2) evaluating the functional consequences of TLR4 inhibition in myotubes derived from human muscle biopsies, and (3) assessing the therapeutic potential of modulating TLR4 signaling in ischemic muscle in a mouse hindlimb ischemia model. Results: TLR4 was found to be expressed in human muscle biopsies, with significant upregulation in samples from patients with CLTI. In vitro studies using cultured human myotubes demonstrated upregulation of TLR4 in ischemia, with activation of the downstream signaling pathway. Inhibition of TLR4 before ischemia was associated with reduced ischemia-induced apoptosis. Upregulation of TLR4 also occurred in ischemia in vivo and TLR4 inhibition was associated with decreased inflammatory cell infiltration and diminished apoptosis in the ischemic limb. Conclusions: TLR4 is upregulated and activated in ischemic skeletal muscle in patients with CLTI. Modulating TLR4 signaling in vitro and in vivo was associated with attenuation of ischemia-induced skeletal muscle damage. This strategy could be explored further for potential clinical application.